LOCUS       sequence-415798-        5298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unfused EcKlenow DNA polymerase (-exo), expressed from CMV or T7 
            promoters. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5298)
  AUTHORS   Ferreira da Silva J, Tou CJ, King EM, Eller ML, Rufino-Ramos D, Ma 
            L, Cromwell CR, Metovic J, Benning FMC, Chao LH, Eichler FS, 
            Kleinstiver BP
  TITLE     Click editing enables programmable genome writing using DNA 
            polymerases and HUH endonucleases.
  JOURNAL   Nat Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x.
  PUBMED    39039307
REFERENCE   2  (bases 1 to 5298)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2024 Jul 22. doi: 10.1038/s41587-024-02324-x."
COMMENT     Sequence Label: Polymerase expression - pCMV-T7-EcKlenow (LM2692)
FEATURES             Location/Qualifiers
     source          1..5298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2284..2304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2313..2330
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2353..2370)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2359..2583
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2654..2670)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2654..2670)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2667..2689)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2678..2694
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2702..2708,2709..2726,2727..2732))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2747..2768
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2885..2902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3056..3644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3136..3155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3815..4606,4607..4675))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4438..4457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4676..4780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4859..4878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5050..5298,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggttatctcc
      421 tacgacaact acgtgaccat cctggacgaa gagacactga aggcttggat cgctaaactg
      481 gaaaaagccc ctgtgttcgc cttcgccacc gccaccgact ccttagataa catcagcgcc
      541 aacctggtgg gcctgtcttt cgccatcgag cctggcgtgg ccgcctacat ccccgtggcc
      601 cacgattacc tggatgcccc agatcaaatc agcagagaga gagcactgga gctcctgaag
      661 cccctgctgg aggacgagaa ggccctgaag gtgggacaga acctgaaata cgatagggga
      721 atcctggcta attacggcat cgagctgaga ggcatcgcct tcgataccat gctggaatct
      781 tacatcctga acagcgtggc cggccgccac gacatggaca gcctggctga acggtggctg
      841 aagcacaaaa ccatcacctt cgaagagatc gccggaaagg gcaagaacca gctgacattt
      901 aatcagatcg cactggaaga agccggcaga tacgccgctg aggacgccga cgtgaccctt
      961 cagctgcacc tgaagatgtg gcccgatctg cagaagcaca agggcccact aaacgtgttc
     1021 gagaacatcg agatgcctct ggtgcccgtc ctgagcagaa tcgagaggaa tggcgtgaag
     1081 atcgacccca aggtgctgca caaccacagc gaggaactga cactgcggct ggccgagctg
     1141 gaaaagaagg cccacgaaat cgccggcgag gagttcaacc tgtctagcac caagcagcta
     1201 caaacaatcc tgttcgagaa gcagggcatt aagcctctga aaaagacacc tggcggagcc
     1261 cccagcacca gcgaagaagt gctggaggaa ctggcgctgg attaccctct gcccaaagtg
     1321 atcctggaat atagaggact ggccaagctg aagtctacat acaccgacaa gctgcctctg
     1381 atgatcaacc ccaagaccgg ccgggtgcat acatcttatc accaggccgt gacagccacc
     1441 ggccggctga gcagcacaga tcctaacctg caaaacatcc cagtgcggaa cgaagagggc
     1501 agaagaatca gacaggcttt cattgcccct gaggactacg tcatcgtgtc tgctgactac
     1561 agccagatag aactgagaat catggcccat ctgtctcggg acaagggcct gctgaccgct
     1621 tttgcagaag gcaaggacat ccacagagcc accgctgccg aagtgttcgg cctgcctctg
     1681 gaaacagtga ccagcgagca aagaaggtcc gccaaggcca ttaacttcgg cctgatctac
     1741 ggcatgagcg cctttggcct ggccagacag ctcaacatcc ctagaaagga ggcccagaaa
     1801 tacatggacc tgtacttcga gcggtacccc ggcgtgctgg agtacatgga acggaccaga
     1861 gcccaggcta aagagcaggg atacgtggaa accctggatg gccggagact gtatctgcct
     1921 gacatcaaga gcagcaacgg cgccagaaga gccgccgctg agagagctgc tatcaacgcc
     1981 cctatgcagg gcaccgccgc cgacatcatc aagcgggcca tgatcgccgt ggacgcctgg
     2041 ctgcaggccg agcagcctag agtgcggatg atcatgcagg tgcacgacga gctggtgttt
     2101 gaggtgcaca aggacgacgt ggatgctgtg gccaagcaga ttcaccagct gatggaaaat
     2161 tgcaccagac tggacgtgcc actgctggtc gaggttggaa gcggagagaa ttgggaccag
     2221 gcccactccg gcggaagctc tggtggcagc aagcggaccg ccgacggctc tgaattcgag
     2281 agccctaaga agaaaagaaa ggtgtgaccg gtcatcatca ccatcaccat tgagtttaaa
     2341 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2401 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2461 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2521 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2581 tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct
     2641 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2701 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     2761 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2821 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2881 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2941 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3001 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3061 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3121 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3181 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3241 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3301 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3361 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3421 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3481 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3541 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3601 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3661 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3721 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3781 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3841 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3901 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgcgaac
     3961 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4021 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4081 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4141 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4201 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4261 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4321 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4381 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4441 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4501 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4561 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4621 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4681 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4741 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4801 cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta
     4861 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     4921 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     4981 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     5041 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     5101 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     5161 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     5221 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     5281 cagtacatca agtgtatc
//
